CC Chemokine Receptor Research Articles

Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5.

CC chemokine receptors 2 and 5 (CCR2 and CCR5) are GPCRs that guide monocytes towards regions of inflammation, thus shaping the inflammatory response. CCR2 & CCR5 have been extensively studied as potential targets in Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH), where antagonizing CCR2 reduces Kupffer cell activation, lowers monocyte infiltration, and prevents liver fibrosis. In the case of pancreatic ductal adenocarcinoma (PDAC), a lethal cancer, CCR2-mediated macrophage infiltration of the tumor microenvironment leads to poor patient survival and increased resistance to chemo-, radiation, and immuno-therapies, thus motivating studies of CCR2 antagonists in combination with these therapies. However, no CCR2 antagonist has yet reached the clinic. Redundancies inherent to the chemokine system make targeting this receptor difficult. Many inhibitors of CCR2 have comparable affinity for CCR5; while dual-selective CCR2/CCR5 antagonists may be indicated for NASH, dual-selectivity hinders their use for PDAC. To guide the design of better CCR2- and CCR5-targeting small-molecule therapeutics, it is necessary to understand the structural basis of antagonist interaction with these highly homologous receptors.

Determination of the Binding Epitope of an Anti-Mouse CCR9 Monoclonal Antibody (C9Mab-24) Using the 1× Alanine and 2× Alanine-Substitution Method

C-C chemokine receptor 9 (CCR9) is a receptor for C-C-chemokine ligand 25 (CCL25). CCR9 is crucial in the chemotaxis of immune cells and inflammatory responses. Moreover, CCR9 is highly expressed in tumors, including several solid tumors and T-cell acute lymphoblastic leukemia. Several preclinical studies have shown that anti-CCR9 monoclonal antibodies (mAbs) exert antitumor activity. Therefore, CCR9 is an attractive target for tumor therapy. In this study, we conducted the epitope mapping of an anti-mouse CCR9 (mCCR9) mAb, C9Mab-24 (rat IgG2a, kappa), using the 1× alanine (1× Ala)- and 2× alanine (2× Ala)-substitution methods via enzyme-linked immunosorbent assay. We first performed the 1× Ala-substitution method using one alanine-substituted peptides of the mCCR9 N-terminus (amino acids 1-19). C9Mab-24 did not recognize two peptides (F14A and F17A), indicating that Phe14 and Phe17 are critical for C9Mab-24-binding to mCCR9. Furthermore, we conducted the 2× Ala-substitution method using two consecutive alanine-substituted peptides of the mCCR9 N-terminus, and showed that C9Mab-24 did not react with four peptides (M13A-F14A, F14A-D15A, D16A-F17A, and F17A-S18A), indicating that 13-MFDDFS-18 is involved in C9Mab-24-binding to mCCR9. Overall, combining, the 1× Ala- or 2× Ala-scanning methods could be useful for understanding for target-antibody interaction.

The dual-function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct mechanisms.

C-C chemokine receptor 2 (CCR2) is a dual-function receptor. Similar to other G protein-coupled chemokine receptors, it promotes monocyte infiltration into tissues in response to the chemokine CCL2, and, like atypical chemokine receptors (ACKRs), it scavenges chemokine from the extracellular environment. CCR2 therefore mediates CCL2-dependent signaling as a G protein-coupled receptor (GPCR) and also limits CCL2 signaling as a scavenger receptor. We investigated the mechanisms underlying CCR2 scavenging, including the involvement of intracellular proteins typically associated with GPCR signaling and internalization. Using CRISPR knockout cell lines, we showed that CCR2 scavenged by constitutively internalizing to remove CCL2 from the extracellular space and recycling back to the cell surface for further rounds of ligand sequestration. This process occurred independently of G proteins, GPCR kinases (GRKs), β-arrestins, and clathrin, which is distinct from other "professional" chemokine scavenger receptors that couple to GRKs, β-arrestins, or both. These findings set the stage for understanding the molecular regulators that determine CCR2 scavenging and may have implications for drug development targeting this therapeutically important receptor.

Genetic code expansion to enable site-specific bioorthogonal labeling of functional G protein-coupled receptors in live cells.

For use in site-specific bioorthogonal labeling of expressed G protein-coupled receptors (GPCRs) in live cells, we developed a luciferase-based reporter assay. The assay was used to compare amber codon suppression efficiency, receptor functionality, and efficiency of different bioorthogonal labeling chemistries. We used the assay system to compare side-by-side the efficiency of incorporation of three different noncanonical amino acids [4-azido-l-phenylalanine (azF), cyclopropene-l-lysine (CpK), and trans-cyclooct-2-en-l-lysine (TCOK)] at three different sites on a GPCR using three different genetic code expansion plasmid systems. As a model GPCR, we engineered an epitope-tagged C-C chemokine receptor 5 (CCR5)-RLuc3 fusion for expression in HEK293T cells. Satisfactory incorporation of azF, CpK, and TCOK into heterologously expressed CCR5 was achieved. We also carried out cell-based calcium mobilization assays to measure the function of the engineered CCR5, and in the same cells, we performed bioorthogonal labeling of the engineered mutants using heterobivalent compounds containing bioorthogonal tethering groups linked to either a small-molecule fluorophore or a peptide. Favorable reaction kinetics of tetrazine-containing compounds with CCR5 harboring TCOK was observed. However, bioorthogonal labeling in live cells of CCR5 harboring CpK with tetrazine-containing compounds using the inverse electron demand Diels-Alder ligation was overall slightly more efficient than other reactions tested.

Distinct phenotype of neutrophil, monocyte, and eosinophil populations indicates altered myelopoiesis in a subset of patients with multiple myeloma.

Hematologic malignancies, including multiple myeloma (MM), promote systemic immune dysregulation resulting in an alteration and increased plasticity of myeloid cell subsets. To determine the heterogeneity of the myeloid cell compartment in the peripheral blood of patients with MM, we performed a detailed investigation of the phenotype and function of myeloid subpopulations. We report that a subset of MM patients exhibits a specific myeloid cell phenotype indicative of altered myelopoiesis characterized by significant changes in the properties of circulating granulocytic, monocytic, and eosinophilic populations. The subset, referred to as MM2, is defined by a markedly elevated level of CD64 (FcγRI) on the surface of circulating neutrophils. Compared to healthy controls or MM1 patients displaying intermediate levels of CD64, neutrophils from MM2 patients exhibit a less differentiated phenotype, low levels of CD10 and CXC chemokine receptor 2 (CXCR2), increased capacity for the production of mitochondrial reactive oxygen species, and an expansion of CD16neg immature neutrophil subset. Classical and patrolling monocytes from MM2 patients express elevated levels of CD64 and activation markers. MM2 eosinophils display lower levels of C-C Chemokine receptor 3 (CCR3), Toll-like receptor 4 (TLR4, CD284), and tissue factor (TF, CD142). The MM2 (CD64high) phenotype is independent of age, race, sex, and treatment type. Characteristic features of the MM2 (CD64high) phenotype are associated with myeloma-defining events including elevated involved/uninvolved immunoglobulin free light chain (FLC) ratio at diagnosis. Detailed characterization of the altered myeloid phenotype in multiple myeloma will likely facilitate the identification of patients with an increased risk of disease progression and open new avenues for the rational design of novel therapeutic approaches.

Establishment of Transgenic Mouse Leukemia Cell Lines Expressing Human CD4/CCR5/CyclinT1 Infected with HIV-1.

Establishing a cross-species animal model of human immunodeficiency virus (HIV) infection is crucial for the study of HIV/acquired immunodeficiency syndrome (AIDS). However, due to the species-specific characteristics of HIV-1, the virus can only infect directly humans and a small number of non-human primates. It cannot directly infect mouse cells across species. A mouse leukemia cell line with high CD4 (clusters of differentiation 4)/CCR5 (CC-chemokine receptor 5)/CyclinT1 expression was constructed in this study. First, CD4/CCR5/CyclinT1 lentiviral vector was used to infect a murine leukemia cell line L1210 to express the receptor CD4, co-receptor CCR5 and tat protein driving factor CyclinT1, which are required to infect L1210 cells with HIV-1. The results of sequencing identification and fluorescence expression showed that the plasmid expressing CD4, CCR5, and CyclinT1 vector was successfully constructed and wrapped as the lentiviral vector. Moreover, it was observed that CD4, CCR5, and CyclinT1 proteins were highly expressed in mouse leukemia cells L1210 compared to empty lentiviral vector-transfected cells. Next, viral entry and replication were demonstrated when HIV-1 RNA was detected in body cells and cultured supernatants. Transgenic mice cells L1210 showed significantly greater content of HIV-1 RNA compared to control L1210 cells. Finally, CEMx174 was infected with cell culture supernatants to clarify that the progeny virus is an active virus with infection ability. HIV-1 RNA was highly expressed in CEMx174 cells. This study made the foundation for future studies evaluating HIV-1 cross-species infection in a murine animal model. The results provided new direction for studies investigating the development of vaccines, antiviral drugs screening, and HIV/AIDS pathogenesis.

Investigation of the CC chemokine receptor 5 (CCR5) delta32 and gene variants in HIV infected patients

CCR5 is an important CC chemokine receptor for the normal entry of Human Immunodeficiency Virus (HIV) in host cells. Mutations in this gene have been linked with delayed HIV infection, therefore, the current case-control study was conducted to identify genetic polymorphism in CCR5 gene in Pakistani population and to evaluate its association with resistance for HIV infections. All regulations of MOOSE and Helsinki Declaration were strictly followed during the entire study. DNA was extracted from the blood samples of HIV positive and HIV negative patients. Extracted DNA was amplified for CCR5 gene by PCR. Amplified product was sequenced to screen out polymorphism mutation, including most prevalent 32 base-pairs deletion through Bioedit. Novel SNP in 5?UTR region was identified and examined by CRYP-SKIP server. None of the studied samples demonstrated any previously reported polymorphisms. CRYP-SKYP server predicted that this polymorphism has no effect on splicing or transcription of CCR5 gene. Genetic Polymorphism of delta32 mutation in CCR5 gene was found in Pakistani individuals. To the best of our knowledge, this is the first report from this region. However, large scale studies should be conducted for extensive view of the association of delta32 mutation in CCR5 gene and resistance for HIV infection.

Monocyte chemotactic protein (MCP)-1 (CCL2) and its receptor (CCR2) are elevated in chronic heart failure facilitating lung monocyte infiltration and differentiation which may contribute to lung fibrosis.

Dyspnea, the cardinal manifestation of chronic heart failure (CHF), may reflect both pulmonary oedema and pulmonary remodeling resulting in tissue stiffening. Emerging evidence suggests that predominance of distinct phenotypes of alveolar and recruited macrophages, designated M1 and M2, may regulate the course of inflammatory tissue repair and remodeling in the lung. In a CHF rat model, we found fibrotic reinforcement of the extracellular matrix with an increase in monocyte chemotactic protein (MCP)-1/CCL2 in bronchoalveolar lavage (BAL), corresponding to a 3-fold increase in recruited macrophages. In this clinical cross sectional study, we aimed to examine potential mediators of leukocyte activation and lung infiltration in parallel BAL and blood from CHF patients compared to non-CHF controls. Mini-BAL and peripheral blood samples were obtained from hospitalized CHF, acute decompensated CHF and non-CHF patients. CHF patients and decompensated CHF patients demonstrated increases from non-CHF patients in BAL MCP-1, as well as the M2 macrophage cytokines interleukin-10 and transforming growth factor-β. BAL and plasma MCP-1 were significantly correlated; however, MCP-1 was 20-fold higher in epithelial lining fluid in BAL, indicative of an alveolar chemotactic gradient. An increase in transglutaminase 2 positive M2 macrophages in parallel with a decrease in the MCP-1 receptor, CC chemokine receptor 2 (CCR2), was apparent in BAL cells of CHF patients compared to non-CHF. These data suggest a pathway of MCP-1 mediated M2 macrophage prevalence in the lungs of CHF patients which may contribute to pulmonary fibrotic remodeling and consequent increased severity of dyspnea.

Mogamulizumab Multimodality Therapy with Systemic Retinoids, Interferon, or Extracorporeal Photopheresis for Advanced Cutaneous T‐Cell Lymphoma

Mogamulizumab is a novel monoclonal antibody designed to target CC chemokine receptor 4 (CCR4), which is expressed by tumor cells in cutaneous T‐cell lymphoma (CTCL). In clinical trials, mogamulizumab monotherapy has demonstrated efficacy in reducing tumor burden in advanced CTCL, particularly in the peripheral blood. In clinical practice at our center, mogamulizumab has been combined with other agents such as systemic interferon, systemic retinoids, and extracorporeal photopheresis for an enhanced synergistic effect. There is limited published research on mogamulizumab combination therapy. This study evaluates the clinical efficacy of mogamulizumab in combination with systemic interferon, retinoids, and/or extracorporeal photopheresis for therapy of CTCL. Nineteen patients on this regimen at our academic center were identified. Treatment response rates, progression‐free survival, duration of response, and adverse effects were characterized by retrospective chart review. All patients on this regimen at our center had an initial partial or complete response. Moreover, responses were durable, and the regimen was well‐tolerated with few grade 3 adverse effects. These results demonstrate the utility of a multimodality approach to the therapy of CTCL with mogamulizumab and warrant further confirmation.

CC-Chemokine Receptor-2 Expression in Osteoblasts Contributes to Cartilage and Bone Damage during Post-Traumatic Osteoarthritis

CC-Chemokine Receptor-2 Expression in Osteoblasts Contributes to Cartilage and Bone Damage during Post-Traumatic Osteoarthritis

Impact of cancer-associated mutations in CC chemokine receptor 2 on receptor function and antagonism

CC chemokine receptor 2 (CCR2), a G protein-coupled receptor, plays a role in many cancer-related processes such as metastasis formation and immunosuppression. Since ∼ 20 % of human cancers contain mutations in G protein-coupled receptors, ten cancer-associated CCR2 mutants obtained from the Genome Data Commons were investigated for their effect on receptor functionality and antagonist binding. Mutations were selected based on either their vicinity to CCR2′s orthosteric or allosteric binding sites or their presence in conserved amino acid motifs. One of the mutant receptors, namely S101P2.63 with a mutation near the orthosteric binding site, did not express on the cell surface. All other studied mutants showed a decrease in or a lack of G protein activation in response to the main endogenous CCR2 ligand CCL2, but no change in potency was observed. Furthermore, INCB3344 and LUF7482 were chosen as representative orthosteric and allosteric antagonists, respectively. No change in potency was observed in a functional assay, but mutations located at F1163.28 impacted orthosteric antagonist binding significantly, while allosteric antagonist binding was abolished for L134Q3.46 and D137N3.49 mutants. As CC chemokine receptor 2 is an attractive drug target in cancer, the negative effect of these mutations on receptor functionality and drugability should be considered in the drug discovery process.

Establishment of a Sensitive Monoclonal Antibody Against Mouse CCR9 (C9Mab-24) for Flow Cytometry

The CC chemokine receptor 9 (CCR9), also known as CD199, is one of chemokine receptors. The CC chemokine ligand 25 (CCL25) is known to be the only ligand for CCR9. The CCR9-CCL25 interaction plays important roles in chemotaxis of lymphocytes and tumor cell migration. Therefore, CCR9-CCL25 axis is a promising target for tumor therapy and diagnosis. In this study, we established a sensitive and specific monoclonal antibody (mAb) against mouse CCR9 (mCCR9) using N-terminal peptide immunization method. The established anti-mCCR9 mAb, C9Mab-24 (rat immunoglobulin [IgG]2a, kappa), reacted with mCCR9-overexpressed Chinese hamster ovary-K1 (CHO/mCCR9) and mCCR9-endogenously expressed cell line, RL2, through flow cytometry. Kinetic analyses using flow cytometry showed that the dissociation constants (KD) of C9Mab-24 for CHO/mCCR9 and RL2 cell lines were 6.0 × 10-9 M and 4.7 × 10-10 M, respectively. Results indicated that C9Mab-24 is useful for detecting mCCR9 through flow cytometry, thereby providing a possibility for targeting mCCR9-expressing cells in vivo experiments.

CC Chemokine Receptor 4 (CCR4) as a Possible New Target for Therapy.

Chemokines and their receptors participate in many biological processes, including the modulation of neuroimmune interactions. Approximately fifty chemokines are distinguished in humans, which are classified into four subfamilies based on the N-terminal conserved cysteine motifs: CXC, CC, C, and CX3C. Chemokines activate specific receptors localized on the surface of various immune and nervous cells. Approximately twenty chemokine receptors have been identified, and each of these receptors is a seven-transmembrane G-protein coupled receptor. Recent studies provide new evidence that CC chemokine receptor 4 (CCR4) is important in the pathogenesis of many diseases, such as diabetes, multiple sclerosis, asthma, dermatitis, and cancer. This review briefly characterizes CCR4 and its ligands (CCL17, CCL22, and CCL2), and their contributions to immunological and neoplastic diseases. The review notes a significant role of CCR4 in nociceptive transmission, especially in painful neuropathy, which accompanies many diseases. The pharmacological blockade of CCR4 seems beneficial because of its pain-relieving effects and its influence on opioid efficacy. The possibilities of using the CCL2/CCL17/CCL22/CCR4 axis as a target in new therapies for many diseases are also discussed.

Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry.

The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-3 (rat IgG2a, kappa), and C3Mab-4 (rat IgG2a, kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C3Mab-3, C3Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1-38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C3Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C3Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C3Mab-3, C3Mab-4, and J073E5.

CC chemokine receptor 2 (CCR2) expression promotes diffuse large B-Cell lymphoma survival and invasion

In recent years, CC chemokine receptor 2 (CCR2) has been found to be involved in tumor growth, angiogenesis, epithelial mesenchymal transition, metastasis, and immune escape. CCR2 overexpression was first identified as a poor prognostic predictor in diffuse large B-cell lymphoma (DLBCL) in our published article, but the mechanisms involved remain unknown. In this work, we collected data from another 138 patients with DLBCL data and verified the CCR2 expression level and its relationship to clinicopathological characteristics. Furthermore, we explored the possible mechanisms via in vitro and in vivo experiments. We showed that CCR2 overexpression was an independent prognostic marker and predicted shorter overall survival (OS) and progression-free survival (PFS) in patients with DLBCL. Blockade of CCR2 expression with a CCR2 antagonist inhibited tumor cell proliferation, migration, and anti-apoptosis ability in vitro by affecting the PI3K/Akt signaling pathway and the p38 MAPK signaling pathway. Furthermore, administration of a CCR2 antagonist decreased tumor growth and dissemination of DLBCL cells and increased survival time in the xenograft model. Our study demonstrates that CCR2 expression plays an important role in the development of DLBCL by stimulating cell proliferation, migration, and anti-apoptosis. Therefore, the inhibition of CCR2 may be a potential target for anticancer therapy in DLBCL.The authors show that CC chemokine receptor 2 (CCR2) overexpression is an independent prognostic marker for diffuse large B-cell lymphoma (DLBCL) and predicts overall survival and progression-free survival in these patients. Blockade of CCR2 signaling with a CCR2 antagonist inhibits tumor cell proliferation, migration, and apoptosis inhibition by activating the PI3K/Akt signaling pathway and inhibiting the p38 MAPK signaling pathway. Furthermore, administration of a CCR2 antagonist decreases tumor growth and dissemination of DLBCL cells and increases survival time in a xenograft model.

Targeting CCL5 signaling attenuates neuroinflammation after seizure.

Epilepsy is a neurological condition that causes unprovoked, recurrent seizures. Accumulating evidence from clinical and experimental studies indicates that neuroinflammation exacerbates seizure activity. We investigated the transcriptional changes occurring in specific brain domains of a seizure mouse model, using 10× Genomics spatial transcriptomics. Differential gene expression and pathway analysis were applied to investigate potential signaling targets for seizure, including CCL5/CCR5 pathway. Maraviroc, an FDA-approved C-C chemokine receptor 5 (CCR5) antagonist, was used to verify the impact of CCL5/CCR5 signaling in seizure mice. We found distinguished regional transcriptome features in the hippocampus of seizure mice. The hippocampus exhibited unique inflammatory gene signatures, including glia activation, apoptosis, and immune response in seizure mice. Especially, we observed notable expression of C-C chemokine ligand 5 (CCL5) throughout the entire seizure hippocampus. Blockade of CCL5/CCR5 signaling via maraviroc prevented microglia activation and neuron degeneration in seizure mice. This study supports the potential of CCL5/CCR5 signaling for targeting neuroinflammation after seizure.

Development of a Novel Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) by N-Terminal Peptide Immunization.

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor family member that is highly expressed in B lymphocytes, certain subsets of effector and memory T cells, and immature dendritic cells. CCR6 has only one chemokine ligand, CCL20. The CCL20-CCR6 axis has been recognized as a therapeutic target for autoimmune diseases and tumor. This study developed specific monoclonal antibodies (mAbs) against mouse CCR6 (mCCR6) using the peptide immunization method. The established anti-mCCR6 mAb, C6Mab-13 (rat IgG1, kappa), reacted with mCCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCCR6), and mCCR6-endogenously expressed P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells in flow cytometry. The dissociation constant (KD) of C6Mab-13 for CHO/mCCR6 cells was determined to be 2.8 × 10-9 M, indicating that C6Mab-13 binds to mCCR6 with high affinity. In summary, C6Mab-13 is useful for detecting mCCR6-expressing cells through flow cytometry.

Development of a Sensitive Anti-Human CCR9 Monoclonal Antibody (C9Mab-11) by N-Terminal Peptide Immunization.

The C-C chemokine receptor 9 (CCR9) belongs to the G-protein-coupled receptor superfamily, and is highly expressed on the T cells and intestinal cells. CCR9 regulates various immune responses by binding to the C-C chemokine ligand, CCL25, and is involved in inflammatory diseases and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR9 is necessary for treatment and diagnosis. In this study, we established a specific anti-human CCR9 (hCCR9) mAb; C9Mab-11 (mouse IgG2a, kappa), using the synthetic peptide immunization method. C9Mab-11 reacted with hCCR9-overexpressed Chinese hamster ovary-K1 (CHO/hCCR9) and hCCR9-endogenously expressed MOLT-4 (human T-lymphoblastic leukemia) cells in flow cytometry. The dissociation constant (KD) of C9Mab-11 for CHO/hCCR9 and MOLT-4 cells were determined to be 1.2 × 10-9 M and 4.9 × 10-10 M, respectively, indicating that C9Mab-11 possesses a high affinity for both exogenously and endogenously hCCR9-expressing cells. Furthermore, C9Mab-11 clearly detected hCCR9 protein in CHO/hCCR9 cells using western blot analysis. In summary, C9Mab-11 can be a useful tool for analyzing hCCR9-related biological responses.

Effective Cell Dose and Functional Attributes of Azercabtagene Zapreleucel (azer-cel; PBCAR0191) Associate with Allogeneic CAR T-Cell Safety and Efficacy in Patients with Relapsed/Refractory B-Cell Lymphoma

Background: Characterization of product cellular attributes contributing to the in vivo expansion and toxicity of allogeneic, CD19-directed chimeric antigen receptor (CAR) T-cell therapy is necessary to optimize efficacy and safety. Using a single step process, the azer-cel CD19 CAR is knocked into the T-cell receptor alpha constant (TRAC) gene locus via a TRAC-specific ARCUS nuclease in activated T cells. Unlike autologous CAR T-cell therapy, all allogeneic CAR T products are cryopreserved. In this phase I study of the allogeneic, CD19-directed CAR T azer-cel, we analyzed post-thaw product attributes and cell composition of the infused product associated with pharmacokinetics (PK), pharmacodynamics (PD), and clinical outcomes (NCT03666000). Methods: Azer-cel was administered as a single infusion to 44 subjects across several dose levels and fludarabine/cyclophosphamide lymphodepletion regimens. CAR T cell peak expansion (Cmax), PK and area under the concentration-time curve (AUC) was assessed by flow cytometry and PD by multiplex cytokine assays. Azer-cel post-thaw cell composition was assessed by flow cytometry (minimally differentiated stem central memory [SCM], CC-chemokine receptor 7 (CCR7)+; differentiated effector memory, CCR7-). Effective cell number infused for any antigenically defined population was estimated using the product of the population percentage and the total post-thaw viable cell number. Overall tumor burden was measured as sum of the products of perpendicular diameters of target lesions and response was evaluated using Lugano 2016 criteria. Responders had either a complete or partial response at Day 28 or later. Relationships to safety events and efficacy were assessed using univariate correlative analyses (variables selected based on Pearson correlation coefficients ρ(rho)>0.35 unless specified, P-value <0.05). Results: Peak azer-cel levels and AUC varied by dose level with median Cmax ranging from 38 cells/mL at DL2 to 6.7x 104 cells/mL at DL4b, whereas median AUC0-28 ranged between 3 x 102 to 5.1x 105(cells/mL*days). Both azer-cel Cmax and AUC correlated strongly with SCM cell dose (P=0.009 & P=0.0026, respectively). Development of cytokine release syndrome (CRS) associated with effective SCM CD8+ cell dose (P<0.05) and total tumor burden (P=0.0087), in addition to Cmax of the cytokines interleukin (IL)-6, TNFα, MIP1α, IL-10 and C-X-C motif chemokine ligand 10 (CXCL10) or Interferon gamma-induced protein 10 (IP10) (P<0.01). Immune effector cell-associated neurotoxicity syndrome (ICANS) correlated with azer-cel Cmax (P=0.035), AUC (P=0.0057), total tumor burden (P=0.004), and effective SCM CAR T cell dose (P<0.05), as well as changes in a distinct set of cytokines that included IL-2, IL-8, IIL-15, IL-6, and IP10 (P<0.05). Grade >3 ICANS correlated strongly with effective SCM CD4+ cell dose (P=0.0015), differentiated CD4+CCR7- cell dose (ρ =0.34, P=0.029) and CD4:CD8 ratio (ρ = 0.32, P= 0.036), in addition to Cmax of IL-6, IL-2 and IP10 (P< 0.01). Overall response to azer-cel treatment negatively correlated with differentiated CD4+ CCR7- cell dose (P=0.04). Conclusions: Post-thaw product composition and effective cell dose are predictive for in vivo expansion potential, CAR-T-related adverse events and response to treatment with azer-cel. This is the first analysis of an allogeneic CD19 CAR T product composition to demonstrate that strategies intended to maximize stem central memory T-cell fraction (CCR7+) while limiting CD4+ CCR7- differentiated fraction may improve safety and efficacy of CAR T therapy.

A Novel Antibody-Drug Conjugate Targeting CCR7 in Hematologic Malignancies

Introduction: CC-chemokine receptor 7 (CCR7) is expressed in lymphoid malignancies and is therefore an attractive target for antibody-drug conjugates (ADCs). JBH492 is a novel, CCR7-targeting ADC, comprised of a humanized anti-CCR7 IgG1 antibody, conjugated in a site-specific manner to the maytansinoid microtubule inhibitor DM4 via a cleavable linker. Methods: CCR7 expression was investigated in human and cynomolgus monkey normal tissue microarrays (TMA) by in situ hybridization (ISH). CCR7 expression across multiple malignancies was investigated by RNA sequencing (RNAseq). The cytotoxic ADC activity of JBH492 was evaluated in vitro in a CellTiter-Glo viability assay. To characterize the impact of the ADC on the CCL19/CCR7 axis, p-ERK was measured as a downstream signaling event of CCL19-mediated CCR7 pathway activation. The in vivo efficacy of JBH492 was investigated in multiple preclinical models of lymphoid malignancies, chosen to cover a range of various CCR7 expression levels, including a panel of 11 DLBCL patient derived xenograft (PDX) models. The safety of JBH492 was assessed in repeat dose toxicity studies conducted in cynomolgus monkeys. Results: RNAseq and flow cytometry reveal high CCR7 expression in lymphoid neoplasms, including chronic lymphocytic leukemia (CLL), B-cell and T-cell non-Hodgkin's lymphoma (NHL), with very limited expression in healthy tissues. Preclinical characterization of JBH492 demonstrates potent, target-dependent cytotoxic activity. In vitro binding studies support avidity-driven binding of JBH492 to CCR7-high tumor cells compared to CCR7-low healthy immune cells. Mechanistic studies of JBH492 suggest a dual mechanism of action, including payload-mediated cytotoxicity, and blockade of ligand-induced CCR7 signaling through antibody antagonistic functionality. In vivo studies showed robust and durable efficacy in multiple clinically relevant lymphoma models. JBH492 achieved PR or CR in 7/11 DLBCL PDX models (response rate ~64%). Nonclinical toxicology studies indicate a favorable safety profile with no notable changes in circulating immune cell populations. Conclusions: Our translational data support the hypothesis that JBH492 may have the potential to become a promising first-in-class ADC for the treatment of CCR7-expressing lymphoid malignancies. The first-in-human clinical trial in patients with refractory/relapsed CLL and non-Hodgkin's lymphoma is currently ongoing (NCT04240704).